Device to expose cells to fluid shear forces and associated systems and methods

ABSTRACT

The present technology relates generally to devices to expose cells to fluid shear forces and associated systems and methods. In particular, several embodiments are directed toward devices to expose cells to fluid shear forces in order to measure changes in internal cell forces. In some embodiments, a fluidic device includes a flow unit configured to induce fluid flow through the device. The device further includes a fluid channel configured to accept a biological sample dispersed on an array of flexible structures. The flow unit can be configured to induce disturbed and/or laminar flow in the fluid channel. The device can further include optical or magnetic detection means configured to measure a degree of deflection of one or more flexible structures in the array.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/552,052, filed Oct. 27, 2011; U.S. Provisional PatentApplication No. 61/645,191, filed May 10, 2012; and U.S. ProvisionalPatent Application No. 61/709,809, filed Oct. 4, 2012, each of which isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with government support under 5T32EB001650 and5R21HL097284 awarded by the National Institutes of Health, andN66001-11-1-4129 awarded by the Space and Naval Warfare Systems Center(SPAWAR). The government has certain rights in the invention.

TECHNICAL FIELD

The present technology relates generally to devices to expose cells tofluid shear forces and associated systems and methods. In particular,several embodiments are directed toward devices to expose cells to fluidshear forces in order measure changes in internal cell forces.

BACKGROUND

Mechanical forces are important modulators of cellular function in manytissues. For example, mechanotransduction responses to laminar ordisturbed flow can strongly affect the ability of endothelial cells tomaintain the barrier between blood and the vessel wall. Laminar flowoccurs in straight vessels and produces a steady shear stress on thecells. Disturbed flow forms downstream of obstructions, bends, orbifurcations, and produces a time-averaged, low shear stress due toeddies in the flow. These flows can activate mechanosensors inendothelial cells that lead to the activation of signaling pathways thataffect cytoskeletal structures. In particular, laminar flow can initiateRho GTPase pathways that cause alignment of actin filaments and assemblyof adherens junctions. Conversely, disturbed flow leads to disorganizedactin, disassembly of adherens junctions, and small gaps betweenadjacent endothelial cells. These structural changes in endothelialcells can strongly affect the integrity of the vascular barrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a partially schematic illustration of a cellular flow chamberconfigured in accordance with embodiments of the technology.

FIG. 1B is an isometric, partially schematic illustration of an array ofmicropost substrates configured for placement in the cellular flowchamber of FIG. 1A in accordance with embodiments of the technology.

FIGS. 2A-2C are illustrations of cells cultured on flat substrates andplaced under static, laminar, or disturbed flow conditions andconfigured in accordance with embodiments of the technology.

FIGS. 2D-2F are rose plots illustrating the orientations of the cells ofFIGS. 2A-2C, respectively, in accordance with embodiments of the presenttechnology.

FIGS. 3A-3C are illustrations of cells cultured on microposts and placedunder static, laminar, or disturbed flow conditions and configured inaccordance with embodiments of the technology.

FIGS. 3D-3F are rose plots illustrating the orientations of the cells ofFIGS. 3A-3C, respectively, in accordance with embodiments of the presenttechnology.

FIG. 4A is an illustration of vector fields of intercellular force inaccordance with embodiments of the technology.

FIG. 4B is a graph illustrating intercellular forces under static,laminar, and disturbed flow conditions in accordance with embodiments ofthe technology.

FIG. 5 is a block diagram illustrating a method of applying shear forceto a biological sample in accordance with embodiments of the technology.

DETAILED DESCRIPTION

The present technology relates generally to devices to expose cells tofluid shear forces and associated systems and methods. In particular,several embodiments are directed toward devices to expose cells to fluidshear forces in order to measure changes in internal cell forces. Insome embodiments, a fluidic device includes a flow unit configured toinduce fluid flow through the device. The device further includes afluid channel configured to accept a biological sample dispersed on anarray of flexible structures. The flow unit can be configured to inducedisturbed and laminar flow in the fluid channel. The device can furtherinclude detection means configured to measure a degree of deflection ofone or more flexible structures in the array.

Specific details of several embodiments of the technology are describedbelow with reference to FIGS. 1-5. Other details describing well-knownstructures and systems often associated with cellular research toolshave not been set forth in the following disclosure to avoidunnecessarily obscuring the description of the various embodiments ofthe technology. Many of the details, dimensions, angles, and otherfeatures shown in the Figures are merely illustrative of particularembodiments of the technology. Accordingly, other embodiments can haveother details, dimensions, angles, and features without departing fromthe spirit or scope of the present technology. A person of ordinaryskill in the art, therefore, will accordingly understand that thetechnology may have other embodiments with additional elements, or thetechnology may have other embodiments without several of the featuresshown and described below with reference to FIGS. 1-5.

FIG. 1A is a partially schematic illustration of a parallel plate fluidflow chamber 100 configured in accordance with embodiments of thetechnology. In several embodiments, the flow chamber 100 is sized forbenchtop use to simulate fluid shear stresses on various cell typesexposed to laminar and dynamic fluid flow in their natural,physiological environment. In some embodiments, at least a portion ofthe flow chamber 100 is constructed of clear acrylic or other suitablematerial.

The chamber 100 can include a main channel 102 through which pumpedfluid flows. In one embodiment, the main channel 102 has a length of 100mm, a width of 20 mm, and a depth of 0.5 mm. The main channel 102 canhave other dimensions in other embodiments. While FIG. 1A illustrates asingle main channel 102, in further embodiments the chamber 100 caninclude a plurality of fluid channels. The main channel 102 isconfigured to accept one or more individual or arrays 110 of microposts.As discussed in further detail below with reference to FIG. 1B, in someembodiments, the array 110 comprises a biological sample that iscultured as monolayers on micropost substrates. In several embodiments,the sample comprises human pulmonary artery endothelial cells, orHPAECs. In further embodiments, the sample comprises whole blood,platelets, tumor cells, protein solutions, fibroblasts, smooth musclecells, cardiomyocytes, white blood cells, red blood cells, or fragmentsthereof. The sample may comprise other types of biological material instill further embodiments. In embodiments with multiple fluid channels,a user can introduce different fluids, biological samples, and/orreagents in the different channels.

FIG. 1B is an isometric, partially schematic illustration of the array110 of micropost substrates configured for placement in the fluid flowchamber 100 of FIG. 1A in accordance with embodiments of the technology.In some embodiments, the array 110 is created by casting a film ofpolydimethylsiloxane (PDMS) or similar material with a 0.25 mm thicknesson a No. 2 glass coverslip. In other embodiments, other materials and/orthicknesses of materials can be used. In some embodiments, theindividual microposts comprise silicon, polymers, metal, or ceramics. Insome embodiments, the microposts or other portion of the chamber 100 issubstantially coated in a non-fouling coating. In a particularembodiment, individual microposts have a diameter from about 1 nm toabout 50 μm and/or an aspect ratio between about 1:1 and about 1:100. Infurther embodiments, the microposts can have other dimensions.

The arrays 110 of microposts can be micromolded into a desiredarrangement. In order to prepare either flat substrates or micropostsfor cell attachment, a binding element (e.g., fibronectin) can beabsorbed onto the surface of a PDMS stamp. The stamps can have nopattern (‘flat stamp’) or an array of positive relief patterns in theshape of a grid of squares (“square stamps”). Once the protein isadsorbed, the stamp can be placed into conformal contact with thesubstrate in order to transfer fibronectin onto the regions of contact.Afterwards, each substrate can be treated with 0.2% Pluronic F127 orother suitable material to ensure that cells adhere to regions where thefibronectin was printed.

As discussed above, in some embodiments, the substrates support HPAECs,which are seeded at confluent densities onto arrays of microposts orflat substrates. In some embodiments, the individual microposts aresubstantially coated with at least one binding element, such asproteins, glycans, polyglycans, glycoproteins, fibrinogen, fibronectin,von Willebrand Factor, collagen, vitronectin, laminin, monoclonalantibodies, polyclonal antibodies, and/or fragments thereof. Cells canbe allowed to spread and reform their adherens junctions for a selectedtime period (e.g., two days) in culture before subjecting them to flowconditions. Microposts can act as elastic, cantilever beams whichdeflect in proportion to the force applied at their tips. Representativedeflection caused by the Flow F applied to the array 110 is illustratedin FIG. 1B as a continuum from micropost base to tip.

Referring to FIGS. 1A and 1B together, in operation, the micropostarrays 110 are placed in the main channel 102 and exposed to static,laminar, and/or disturbed flow conditions for a selected time period.For example, in some embodiments, the substrate arrays 110 with HPAECmonolayers can be placed inside the chamber 100 and shear force can beapplied for the selected time period. In one embodiment, the shear forceis applied continuously for 14 hours. In other embodiments, the force isapplied intermittently or for more or less than 14 hours.

A flow rate (e.g., a steady flow rate of 2 mL) can be produced by a flowunit (e.g., a pump) 104 that is connected to the flow chamber 100 andconfigured to recirculate the media through the chamber 100 in thedirection of the flow arrows. In a particular embodiment, the flow unit104 is capable of introducing the shear stress from about −10 kPa toabout 10 kPa. The flow unit 104 can comprise a positive displacementpump, a piezoelectric pump, a partial vacuum, a diaphragm pump, aperistaltic pump, a hydrostatic pump, or another device. The fluidpassing through the chamber 100 can be any type of fluid configured toinduce the desired shear stress on the array 110. In some embodiments,the fluid contains a drug, reagent, nucleotide, protein, or a materialthat can cause the cells bound to the microposts to contract.

A chamber of air 106 at the entrance of the channel 102 can damp thepulsatile flow so that a steady flow rate is produced in the channel102. The air chamber 106 can be either upstream or downstream of thearray 110. A backward-facing step 108 in the channel 102 can produce aregion of disturbed flow 114 downstream from the step 108. In aparticular embodiment, the step 108 is 0.25 mm tall. Flow in thedisturbed flow region 114 can have separation in its fluid stream lines,stagnation points, and regions of reversal in the direction of flow. Alaminar flow region 112 can occur further downstream from the region ofdisturbed flow 114. In further embodiments, the disturbed flow region114 and laminar flow region 112 can be in other positions relative toone another or relative to the main channel 102. In still furtherembodiments, only one of disturbed flow or laminar flow is produced.

Changes in traction forces, intercellular forces, and adherens junctionscan be measured to understand the role of tension at the cell-cellinterface in regulating the endothelial barrier. For example, the degreeof effect from the flow can be optically determined More specifically,to measure the deflection of a micropost, the difference between theposition of its tip and base can be analyzed from fluorescent imagestaken at the top and bottom of the arrays 110. The magnitude anddirection of each traction force (F) can be computed from the deflection(δ) through the relationship:

$F = {\frac{3\pi\;{ED}^{4}}{64L^{3}}\delta}$

The length L and diameter D of the microposts in the array can bemeasured using a scanning electron microscope. Young's modulus of PDMS(E=2.5 MPa) can be determined by tensile testing. Cytoskeletal tensioncan be assessed by computing the average traction force per monolayer.Intercellular forces can be determined by the vector sum of the tractionforces on a cell in a monolayer. Intercellular tension can be measuredby the average intercellular force for cells within a monolayer.

In further embodiments, deflection can be determined by magneticdetection means. For example, magnetic nanowires can be embedded intomicroposts to apply external forces to cells. In some embodiments, thenanowires comprise cobalt or nickel wires grown by electrochemicaldeposition in the pores of a template. The magnetic microposts can beactuated by applying a uniform field (e.g., in a direction perpendicularto the long axis of the posts). The induced magnetic torque on thenanowire causes the magnetic posts to deflect; the field thereby appliesnanonewtons of force to the biological sample. The nanowires can becomerotated by the deflection of the posts. Micropost deflection can bedetected using a Hall probe placed adjacent to (e.g., underneath) themagnetized array 110.

FIGS. 2A-2C are representative immunofluorescent images of HPAECscultured on flat substrates and placed under (A) static, (B) laminar, or(C) disturbed flow conditions for a selected time period (e.g., 14hours). FIGS. 2D-2F are rose plots illustrating the orientations of theHPAECs of FIGS. 2A-2C, respectively. Endothelial cells can align theiractin filaments in the direction of shear flow on flat substrates.HPAECs grown on flat substrates can align their actin filaments parallelto the direction of laminar flow, whereas under static and disturbedflow, their actin may have no preferential alignment.

FIGS. 3A-3C are representative illustrations of HPAECs cultured onmicroposts and placed under static, laminar, or disturbed flowconditions and configured in accordance with embodiments of thetechnology. FIGS. 3D-3F are rose plots illustrating the orientations ofHPAECs of FIGS. 3A-3C, respectively. Similar to the cells describedabove with reference to FIGS. 2A-2F, actin filaments are orientedpredominately in the direction of laminar flow. This does not hold truefor static or disturbed flow. There is, however, a strong degree ofactin alignment along the 0°, 90°, 180°, and 270° directions for allflow conditions, which can match the orthogonal arrangement of themicroposts in the sample array (e.g., the array 110 illustrated in FIG.1B). These alignment results are due to focal adhesions that can form atthe microposts and therefore help to confine actin filaments betweenadjacent posts. However, laminar flow can create a stronger degree ofalignment of actin in the direction of flow despite the strong influenceof the arrangement of the microposts.

FIG. 4A is an illustration of vector fields of intercellular force for arepresentative HPAEC monolayer exposed to shear force for a selectedtime period (e.g., 14 hours) in accordance with embodiments of thetechnology. FIG. 4B is a graph illustrating representative intercellularforces calculated by the vector sum of the tugging forces acting on acell under static (S), laminar (L), and disturbed (D) flow in accordancewith embodiments of the technology. Referring to FIGS. 4A and 4Btogether, a shear force testing device (such as the fluid flow device)can provide significant information regarding intercellular force. Forexample, vector fields of intercellular force illustrate that cells withlarge intercellular forces acting on them are located not only at theperimeter, but within the interior of the monolayer as well.Cytoskeletal tension can be transmitted as traction forces at themicroposts, but also as intercellular forces between adjacent cellswithin a monolayer. The average intercellular force may be higher forlaminar flow as compared to static and disturbed flow conditions.Intercellular forces can increase under laminar flow and promoteadherens junction assembly.

FIG. 5 is a block diagram illustrating a method 500 of applying shearforce to a biological sample in accordance with embodiments of thetechnology. In some embodiments, the method 500 includes placingmicroposts in a fluid flow device, 510. As discussed above, themicroposts can have a biological sample bound thereto. In particularembodiments, the biological sample comprises HPAECs.

The method 500 further includes flowing fluid over the microposts 520.The fluid can be induced in movement by a pump or other device. Thismethod of flowing fluid can be accomplished in a fluid flow chamber suchas the chamber described above with reference to FIG. 1A. In severalembodiments, flowing fluid over the microposts comprises inducinglaminar and disturbed flow conditions. In some embodiments, these flowconditions can be induced by flowing the fluid over a backward-facingstep in the fluid flow chamber, wherein the laminar and disturbed flowoccur downstream of the step. In further embodiments, the method caninclude placing an air chamber in the fluid flow chamber to induce asteady flow rate. The method 500 can further include measuring thedeflection of the microposts, 540.

The technology disclosed herein offers several advantages over existingsystems. For example, by using the approach of inducing shear flow overa micropost array, a quantitative image analysis can be performed todemonstrate that mechanotransduction of flow directly affects theintercellular tension in a monolayer, which coincides with the assemblyof cell-cell contacts between adjacent cells. The systems disclosedherein can be used as a research tool to investigate numerous aspects ofcellular structure and behavior in various environmental conditions. Forexample, the systems can be used to demonstrate that laminar flow cancause a rise in cytoskeletal tension that increases traction forces andintercellular forces and promotes the assembly of adherens junctions.Disturbed flow can be found to weaken cellular forces and cause adherensjunction disassembly.

From the foregoing it will be appreciated that, although specificembodiments of the technology have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the technology. Further, certain aspects of thenew technology described in the context of particular embodiments may becombined or eliminated in other embodiments. Moreover, while advantagesassociated with certain embodiments of the technology have beendescribed in the context of those embodiments, other embodiments mayalso exhibit such advantages, and not all embodiments need necessarilyexhibit such advantages to fall within the scope of the technology.Accordingly, the disclosure and associated technology can encompassother embodiments not expressly shown or described herein. Thus, thedisclosure is not limited except as by the appended claims.

We claim:
 1. An analytic method, comprising: placing a plurality offlexible microposts in a fluid flow chamber, wherein a plurality ofcells is dispersed over at least two of the plurality of microposts;flowing fluid over the microposts, wherein flowing fluid includesinducing laminar and disturbed flow to the plurality of cells; andmeasuring a deflection of the microposts.
 2. The method of claim 1wherein inducing laminar and disturbed flow comprises flowing fluid overa backward-facing step in the fluid flow chamber, wherein the laminarand disturbed flow occur downstream of the step.
 3. The method of claim1 wherein placing the plurality of microposts includes placingmicroposts having human pulmonary artery endothelial cells boundthereto.
 4. The method of claim 1, further comprising placing an airchamber in the fluid flow chamber to induce a steady flow rate.
 5. Themethod of claim 1 wherein flowing fluid over the microposts comprisesflowing a fluid containing at least one of a drug, reagent, nucleotide,protein.
 6. The method of claim 1, further comprising determining anintercellular tension between at least two of the plurality of cellsbased on the measured deflection.
 7. The method of claim 1, wherein atleast one of the plurality of cells is in simultaneous, direct contactwith two of the microposts.
 8. The method of claim 1, further comprisingseeding the plurality of cells over and across the top surfaces of themicroposts.